The Aryl Hydrocarbon Receptor (AhR) is a ligand-activated transcription factor. Upon binding to a ligand, the AhR translocates from the cytosol to the nucleus, where it mediates the transcription of a wide range of gene targets. Aside from the AhR's role as the mediator of toxicity of various dioxins, recent studies have identified roles for the AhR in apoptosis, cell cycle regulation, and tumor suppression, all of which make the AhR a potentially attractive molecular target for development of novel cancer therapeutics. We hypothesize that certain selective AhR modulators (SAhRMs) are capable of initiating biological responses that should prove useful for prevention and/or treatment of hepatocellular carcinoma. We propose to identify SAhRMs by in silico structure-based Virtual Ligand Screening (VLS) coupled with cell- based AhR transcriptional activation assays. SAhRMs will be screened further for their ability to induce AhR- dependent cell death and/or inhibition of cancer cell proliferation. The availability of numerous well- characterized AhR expressing and non-expressing cell culture models in our lab will enable us to evaluate the functional consequences of AhR activation and the anticancer effects of sAhRMs in hepatoma cells. The Specific Aims proposed in this application are described below. 1. Identify novel AhR modulators that have the potential for treatment of cancer We will use in silico structure-based VLS for identifying novel alternative ligands of the AhR. VLS utilizes a three dimensional structure of a protein or homology model to identify potential binders of a target protein. The Per-Arnt-Sim (PAS)-B domain of AhR is essential for ligand binding. As the AhR ligand binding pocket is not yet crystallized, we have generated and refined mouse and human AhR structural models based on the AhR's homology with existing PAS protein family members whose three-dimensional structures have been solved. These homology models of the AhR will be used to screen both synthetic and natural compound databases by VLS to identify potential binders of the AhR. The effects of the newly identified compounds on AhR activation will be tested by AhR-dependent reporter assays and evaluation of AhR- dependent induction of endogenous target genes. Preliminary screening of a compound library by VLS using the AhR PAS domain homology model described above has already resulted in the identification of some compounds of interest including the clinically used antiandrogen flutamide that promoted transcriptional activation of AhR, induction of AhR- regulated target genes and AhR-dependent inhibition of cancer cell growth. These results strongly support of the feasibility of this exploratory research. 2. Test the compounds identified by VLS for AhR selectivity and potential anticancer effects The compounds identified by VLS will be tested for their ability to directly bind to the AhR. Molecules that are able to bind to the AhR will be tested for their effects on AhR DNA binding and inducing AhR-specific transcriptional activity in liver cancer cells. AhR ligands that induce cell death and/or antiproliferative effects in an AhR-dependent manner in liver cancer cells will be prioritized for further evaluation. 3. Shortlist compounds for future in vivo testing by gene expression profiling We will compare the gene expression induced by SAhRMs to distinguish them from the transcriptional program induced by the classical AhR ligands such as TCDD using human and mouse microarrays. Compounds with expression profiles that differ significantly from that of TCDD, such as our initial lead compounds (flutamide and C-24), and that induce growth inhibition/apoptosis via AhR will be given priority for future in vivo studies. SAhRMs with gene expression profiles highly similar to that of TCDD will not be pursued as anti-cancer agents. The proposed studies are timely and important in the context of developing therapeutic intervention for the prevention and/or treatment of hepatoma via an intriguing molecular target, AhR. Excitingly, the SAhRMs that will be identified by these studies may also be effective against other cancers. We will evaluate some of short-listed SAhRMs for AhR-dependent inhibition of tumor growth in mouse xenografts tumor studies in the future.